The STE20-like protein kinases, GCKR and GCK, are mammalian serine/threonine protein kinases that belong to a subfamily of protein kinases, that also includes GLK, HPK1, NIK, and MST1 and -2. They are characterized by an N-terminal kinase domain related to the yeast STE20 protein kinase and by a large C- terminal regulatory domain. In addition, GCK, GCKR, GLK, HPK1, and NIK are able to activate the stress-activated protein kinase (SAPK, also referred to as Jun kinase or JNK) pathway. Since tumor necrosis factor (TNF) is a major activator of the SAPK pathway, we have examined whether GCK and/or GCKR are important in TNF-induced SAPK activation. TNF is a pleiotropic cytokine, which plays a major role in inflammation. The type 1 receptor for TNF (TNFR1) recruits a complex of proteins that activates downstream signaling pathways; one of those proteins is TRAF2. We have found that both TNF and TRAF2 are potent activators of GCKR and to a lesser extent GCK. In addition, a dominant negative form of TRAF2 blocks TNF-induced GCKR activation. Both a dominant negative form of GCKR and a GCKR antisense construct significantly impaired TNF- and TRAF2-induced SAPK activation, directly implicating GCKR as a downstream effector of TNF. In contrast, the GCKR dominant negative did not impair TNF- or TRAF2-induced NF-kB activation. These results indicate that GCKR is a major mediator of TNF-induced SAPK activation, but not NF-kB activation. They also indicate that there is a bifurcation in the TNF-signaling pathway that leads to SAPK and NF-kB activation, which is below the level of TRAF2 and above the level of GCKR activation. We have extended these observations to show that both GCKR and GCK directly interact with TRAF2. This interaction requires the C-terminal 150 amino acids of both GCK and GCKR and the C-terminus of TRAF2. Furthermore, the C- terminal portion of GCKR (amino acids 386-846) directly co- immunoprecipitates with the C-terminal portion of TRAF2 (amino acids 272-501). While the C-terminus of TRAF2 is required for the interaction with TRAF2, the N-terminus is required for GCKR activation and SAPK activation. Finally, we have shown that TNF- signaling results in the recruitment of GCKR to TRAF2. Thus, the mechanism of TNF-induced SAPK activation is a TRAF2-mediated recruitment of GCKR to the TNF receptor complex and the subsequent activation of GCKR. Whether GCK can play a similar role still needs to be resolved. We have continued our study of the mechanism by which the BCR- Abl oncogene activates the SAPK pathway. This is important because activation of the SAPK pathway has been implicated in the transforming properties of BCR-Abl. We have shown that BCR- Abl tyrosine phosphorylates GCKR, associates with GCKR, and activates GCKR. The activation of GCKR, however, does not depend upon its tyrosine phosphorylation, but rather upon Ras activation. A dominant negative form of Ras blocks both BCR-Abl induced SAPK activation and its induction of GCKR activation. A dominant negative form of GCKR and a GCKR antisense construct block Bcr-Abl induced SAPK activation, directly implicating GCKR in BCR-Abl mediated SAPK activation. We have also studied several Bcr-Abl mutants, all of which are impaired for SAPK activation and for GCKR activation. Finally, we have shown that GCKR is tyrosine phosphorylated and activated in Bcr-Abl positive cell lines derived from patients with chronic myelogenous leukemia. Thus, GCKR may be a major target of Bcr- Abl and contribute to its transforming activity. Recently, we have observed that the tyrosine kinase Pyk2, which has been implicated in chemokine and antigen receptor signaling in lymphocytes, activates GCK. A dominant negative form of GCK blocks Pyk2 induced SAPK. Thus, GCK may be a downstream target of Pyk2.